Toxic effect of triphenyltin on Lemna polyrhiza.

код для вставки на сайт или в блог

ссылки на документ

APPLIED ORGANOMETALLIC CHEMISTRY
Appl. Organometal. Chem. 2005; 19: 807–810
Speciation Analysis and
Published online 9 May 2005 in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/aoc.893
Environment
Toxic effect of triphenyltin on Lemna polyrhiza
Zhihui Song1 * and Guolan Huang2
1
College of Material and Environmental Science, Qingdao University of Science and Technology, Qingdao 266042, People’s Republic
of China
2
College of Environmental Science and Engineering, Nankai University, Tianjin 300071, People’s Republic of China
Received 22 July 2004; Revised 22 December 2004; Accepted 23 December 2004
Little data about toxic effect of triphenyltin (TPT) on aquatic plants is available. The purpose of this
paper is to study the toxic effect of TPT on duckweed, Lemna polyrhiza, and the bioconcentration
factor of TPT by Lemna polyrhiza. At 5 µg/l concentration TPT treatment, a toxic effect on growth of
Lemna polyrhiza appeared. The 8 day IC50 of TPT to Lemna polyrhiza was 19.22 µg/l. TPT stimulated
peroxidase activity and nitrate reductase activity at 2 and 5 µg/l. TPT reduced chloroplast activity of
Lemna polyrhiza at 2 and 5 µg/l. Bioconcentration factors of TPT for Lemna polyrhiza were 4.3 and
10.9 at 2 and 5 µg/l, respectively. Copyright  2005 John Wiley & Sons, Ltd.
KEYWORDS: triphenyltin; Lemna polyrhiza; bioconcentration factor; toxicity
INTRODUCTION
Organotin compounds are used in a variety of consumer
and industrial products including marine antifouling paints,
agricultural pesticides, wood preservatives and plastic
stabilizers. It is widely accepted that antifouling paints are
the most important contributors of organotin compounds to
the marine environment, where they have been responsible
for many deleterious effects on non-target aquatic life.1
There are many sources of contamination by organotin
compounds in coastal areas, with high levels of organotin
compounds observed in marinas, moorings and near vessel
repair facilities. Triphenyltin (TPT) was found in sediment,
plankton and mussels from the port of Osaka and Otsuchi
Bay.2 Concentrations of TPT in sediments from four sites in
the Göta älv Estuary, Southwest Sweden, ranged from 1.5
to 71 ng/g dry weight (d.w.).3 Fish (blue gill, largemouth
bass and channel catfish) from a pond near a pecan orchard
in central Georgia (USA), which had been sprayed with
commercial TPT hydroxide mixtures, contained TPT as well
as diphenyltin (DPT) and monophenyltin (MPT).4 TPT and
triphenyltin (TBT) are known to be immunotoxic and cause
renal and hepatic damage; the relative order of organotin
toxicity in astrocyte cultures is TPT > TBT.5 Organotins (TPT
and TBT) directly inactivate cytochrome P-450 because of
*Correspondence to: Zhihui Song, College of Material and Environmental Science, Qingdao University of Science and Technology,
Qingdao 266042, People’s Republic of China.
E-mail: songhuey@sina.com
Contract/grant sponsor: National Natural Science Foundation of
China; Contract/grant number: 29777010.
interaction with critical sulfhydryl groups of the hemoprotein,
and TBT is metabolized more readily than TPT in rat, hamster
and human.6 The IC50 s of TPT to hepatic glutathione Stransferase activity in marine fish Siganus canaliculatus and
Sparus sarba are 10 and 28 µM, respectively.7 Three types of
hybrid catfish (Clarias gariepinus, Clarias macrocephalus) cell
culture were established to screen toxicity of TPT.8 There is
little information on the effect of TPT on fresh water aquatic
plants. Few reports are available on the bioconcentration of
TPT by aquatic plants. The purpose of this work was to study
the toxic effect of TPT on duckweed, Lemna polyrhiza, and the
bioconcentration factor of TPT by Lemna polyrhiza.
MATERIAL AND METHODS
Duckweed (Lemna polyrhiza) was collected from Weijin
River in Tianjin city. TPT and other toxic pollutants were not detected in the river. Lemna polyrhiza
was cultured in Hoagland medium in the laboratory for 2 weeks. The Hoagland medium consisted
of 0.303 g/l KNO3 , 0.222 g/l CaCl2 , 0.246 g/l MgSO4 · 7H2 O,
0.686 g/l KH2 PO4 , 0.010 g/l FeSO4 , 0.200 g/l EDTA and 2 ml
soil extraction solution in 1 l distilled water. One healthy
Lemna polyrhiza plant was selected for culture in new
Hoagland medium. When the amount of plant was sufficient,
healthy Lemna polyrhiza was the tested.
The IC50 test was conducted in a 250 ml beaker with 200 ml
test solution. Four concentrations of TPT and a control (0, 5,
10, 25, 50unsSn µg/l in Hoagland medium) were used; each
Copyright  2005 John Wiley & Sons, Ltd.
808
Speciation Analysis and Environment
Z. Song and G. Huang
test was repeated three times. Thirteen fronds (leaf blade) of
Lemna polyrhiza were put in to each beaker and covered with
glass. The Beaker was lit at 4000 lx for the entire time at 25 ◦ C.
The test lasted for 8 days.
Every 2 days, the test solution was renewed and the
number of fronds in each beaker was counted. At the end
of the test, whole fronds in each beaker were homogenized
with 10 ml acetone to extract chlorophyll. The extraction
solution was put into a refrigerator for 96 h at 4 ◦ C. Then the
solution was centrifuged at 3000 g for 15 min. The absorbance
of the clear solution was determined at 663 and 64.5 nm.
The chlorophyll content was calculated as follows:
Figure 1. Effect of TPT on the growth of Lemna polyrhiza.
Cchl = 20.2A645 + 8.02A663
(1)
Table 1. Chlorophyll content in Lemna polyrhiza with TPT
treatment at the end of the test
Growth inhibition was calculated as follows:
V = ln(Nt /N0 )/t
I = (V0 − Vt )/V0 × 100%
(2)
(3)
where Cchl is the chlorophyll content, A645 and A663 values of
absorbance of clear solution at 645 and 663 nm, respectively,
V the growth rate, N the number of fronds of Lemna polyrhiza,
or chlorophyll content (Cchl ), and Nt and N0 are N at time
t and initial time. V0 is the growth rate of control, Vt the
growth rate of treatment test at time t and I is the growth
inhibition. I was regressed with values of the logarithms of
TPT concentrations so as to calculate the IC50 value.
The bioconcentration factor (BCF) test was conducted
using a 500 ml beaker containing 400 ml test solution. Two
concentrations of TPT and a control (0, 2, 5 Sn µg/l in
Hoagland medium) were used; each test was repeated three
times. Beakers were lighted at 4000Lx for the entire time at
25 C. The test lasted 8 days.
The test solution was renewed every 2 days and the number
of fronds in each beaker counted. An of 2 mL test solution in
each beaker was used to determine nitrate reductase activity
every 2 days. At the end of the test, five, 10 or more fronds of
Lemna polyrhiza were used to determined chlorophyll content;
five, 10 or more fronds were used to determine sugar content,
10, 20 or more fronds were used to determine peroxidase
activity; and 20, 30 or more fronds were used to determine
chloroplast activity. The remaining fronds in each beaker
were used to determined TPT content.
The methods of determining and calculating nitrate
reductase activity, peroxidase activity, chloroplast activity
and TPT content in duckweed have been published.9
RESULTS AND DISCUSSIONS
The effects of TPT on growth, chlorophyll content, nitrate
reductase activity, peroxidase activity, chloroplast activity
Copyright  2005 John Wiley & Sons, Ltd.
Concentration of TPT
(µg/l)
0
Chlorophyll content in
fronds of Lemna polyrhiza
(mg/g)
5
10
25
50
0.902 0.815 0.582 0.534 0.378
Figure 2. Effect of TPT on the growth of Lemna polyrhiza at
low concentration.
and TPT content in the test solution are shown in Figs 1–6
and Table 1.
From Figs 1 and 2, the toxic effect of TPT on Lemna polyrhiza
appeared at 2 µg/l concentration. At 5 µg/l TPT the growth
rate of Lemna polyrhiza was 88% of the control. At 50 µg/l
TPT, the growth rate of Lemna polyrhiza was 10% of the
control, and the fronds of Lemna polyrhiza became yellow. At
the end of the test, chlorophyll content in fronds of Lemna
polyrhiza exposed to 50 µg/l TPT was 42% of the control.
The 8 day IC50 of TPT on Lemna polyrhiza frond number
was 19.22 µg/l. For chlorophyll content, the 8 day IC50 of
TPT on Lemna polyrhiza was 5.76 µg/l. This showed that
chlorophyll content was more sensitive to TPT than frond
number as an endpoint parameter. Values of 8 day IC50 of
Appl. Organometal. Chem. 2005; 19: 807–810
Speciation Analysis and Environment
Figure 3. Effect of PCP on the nitrate reductase activity of
Lemna polyrhiza.
Figure 4. Effect of TPT on the peroxidase activity of
Lemna polyrhiza.
Figure 5.
Effect of TPT on chloroplast activity of
Lemna polyrhiza.
TPT for Spirulina subsalsa were 15.63 and 9.38 µg/l for growth
rate and chlorophyll content, respectively.10 The chlorophyll
content of the freshwater alga Scenedesmus quadricauda with
10 µg/l TPT treatment was 28% of the control.11 These data
suggest that Lemna polyrhiza was the same sensitivity as bluegreen algae and is less sensitive than green alga to TPT.
Copyright  2005 John Wiley & Sons, Ltd.
Toxic effect of triphenyltin on Lemna polyrhiza
Figure 6. TPT concentration in the test solution.
The results suggest that TPT can decrease the chlorophyll
content of plants, which further damages photosynthesis the
48 h EC50 value for TPT in Daphnia magna was 10.2 µg/l,12
which suggests that Lemna polyrhiza is less sensitive to TPT
than Daphnia magna.
From Fig. 3, nitrate reductase activity increased at low TPT
concentration. Values of nitrate reductase activity were 2.3
and 4.8 times the control at 2 and 5 µg/l TPT, respectively.
This showed that, at low concentration, TPT stimulates nitrate
reductase activity. Nitrogen is an important nutrition element
for plant survival. The abnormal nitrate reductase activity
stimulated by TPT will cause a nitrogen metabolism disorder,
leading to plant death.
Figure 4 shows that the peroxidase activity of Lemna
polyrhiza increased at low TPT concentration. The values
of peroxidase activity were 1.6 and 2.1 times the control,
respectively. Peroxidase is one type of detoxification
enzyme system. Higher peroxidase activity indicated a
detoxification enzyme system working to reduce the
damage from pollutants. Arochlor 1254 (a commercial
mixture of polychlorinated biphenyl congeners) causes
the Lingulodinium polyedrum (dinoflagellate) cells to exhibit
increased ascorbate peroxidase activity (50%).13 Human
glutathione peroxidase activity increased with chlorpyrifosethl (an organophosphate insecticide) at low concentrations.14
However, peroxidase activity at too high or low average
levels will induce the detoxification of plants. The peroxidase
activity of Lemna minor, another species of duckweed, at 2
and 5 µg/l TPT is slightly higher than the control.9 These data
show that TPT, like other pollutants, can stimulate plants
peroxidase activity.
Figure 5 shows the absorbance change curves at 620 nm in
the chloroplast activity determining test. The control curve
decreases more rapidly than that with 2 and 5 µg/l TPT
treatment. The values of chloroplast activity of Lemna polyrhiza
at 2 and 5 µg/l TPT treatment are 33.8 and 13.6% of control,
respectively. However, at the end of the test, chlorophyll
contents of Lemna polyrhiza at 2 and 5 µg/l TPT treatment
were nearly the same as the control. This implies that
chlorophyll content is a perfect indicator of photosynthesis
Appl. Organometal. Chem. 2005; 19: 807–810
809
810
Z. Song and G. Huang
activity. The toxic effect of pollutants on photosynthesis
may appear before chlorophyll content changes. TPT affects
intersystem electron transport in uncoupled chloroplasts,
and uncoupled whole chain electron transport in the
presence of methyl viologen under saturating illumination is
inhibited by 75% at TPT concentrations >80 µM, with a halfmaximal effect at about 20 µM.15 The pollutant can destroy
the chloroplast construct without reducing the chlorophyll
content. Photosynthesis cannot work without the whole
physiological structure, although chlorophyll content is not
changed by the pollutant.
From Table 1, the sugar contents at 2 and 5 µg/l TPT
treatments are 49 and 13% of the control, respectively. Sugar
indicates the assimilation status of plant. The chloroplast is
damaged and the assimilation at sugar is broken down. This
result is like the effect of Cd on wheat.16
From Fig. 6, TPT concentration in the test solution was
nearly stable at the end of test. This suggests that the uptake
TPT from the test solution by Lemna polyrhiza is balanced.
The BCFs of TPT for Lemna polyrhiza were 4.3 and 10.9 at
2 and 5 µg/l TPT, respectively. TPT can be bioconcentrated
by benthic organisms, and at higher trophic levels in the
food chain biodegradation products of TPT were not found.17
Microbial degradation of radio labeled TPT in soil or sediment
samples was slow, with only 5% degradation during a 14day incubation period.4 The concentrations of TPT in fish
muscle from rivers and sea areas in Osaka, Japan were in the
range 0.001–0.130 mg/kg wet weight. The concentrations
of TPT were highest in fish liver, and relatively high
concentrations of TPT were found in heart and brain.18 TPT
BCFs at pH 8 were 2200, 680 and 190 for Thymallus thymallus,
Chironomus riparius and Daphnia magna, respectively.19 TPT
was detected in Crassostrea. gigas with concentrations up
to 678 ng/g from the Chinhae Bay System, Korea.20 In the
foodchain of a shallow freshwater lake in the Netherlands,
zebra mussels, eel, roach, bream, pike, perch, pike perch
and cormorant showed high levels of organotin compounds.
At the lower trophic levels, phenyltin concentrations were
high in benthic species, and at the higher trophic levels, high
net bioaccumulation resulted in high TPT concentrations.21
Concentration of TPT in milkfish flesh in a brackish water
pond was about 230 ng TPT/g wet tissue.22 These means
that TPT can be bioconcentrated by animals and plants,
and biodegradation is difficult. TPT could be transported
to higher trophic level organisms through the food chain,
including duckweed, which could lead to greater toxicity of
TPT to more organisms.
Dunaliella tertiolecta was exposed to TPT; swollen mitochondria were observed and disruption of the thylakoid
membranes of the chloroplast was also observed. It is suggested that inhibition of respiration and photosynthesis
metabolic processes could take place before structural damage
to the responsible organelles is observed.23 The external pectin
theca, the limiting membrane and inter-photosynthetically
active lamellae in the Spirulina subsalsa cell were the targets
that were easily damaged by TPT.24 Those data suggest that
Copyright  2005 John Wiley & Sons, Ltd.
Speciation Analysis and Environment
TPT reduces photosynthetic activity by damaging the physiological structure.
CONCLUSIONS
At 5 µg/l TPT concentration treatment, a toxic effect on the
growth of Lemna polyrhiza appeared. The growth rate of Lemna
polyrhiza decreased with increasing TPT concentration. The
nitrate activity and peroxidase activity of Lemna polyrhiza
increased at 2 and 5 µg/l TPT concentration. The chloroplast
activity Lemna polyrhiza was significantly decreased by TPT
treatment. BCFs of TPT by Lemna polyrhiza were 4.3 and 10.9
at 2 and 5 µg/l concentrations.
Acknowledgments
This research was supported by the National Natural Foundation
Science of China (grant no. 29777010).
REFERENCES
1. Morcillo Y, Borghi V, Porte C. Arch. Environ. Contam. Toxicol.
1997; 32: 198.
2. Harino H, Fukushima M, Yamamoto Y, Kawai S, Miyazaki N.
Arch. Environ. Contam. Toxicol. 1998; 35: 558.
3. Brack K. Water Air, Soil Pollut. 2002; 135: 131.
4. Kannan K, Lee RF. Environ. Toxicol. Chem. 1996; 15: 1492.
5. Karpiak VC, Eyer CL. Cell Biol. Toxicol. 1999; 15: 261.
6. Ohhira S, Watanabe M, Matsui H. Arch. Toxicol. 2003; 77: 138.
7. Al-Ghais SM, Ali B. Bull. Environ. Contam. Toxicol. 1999; 62: 207.
8. Visoottiviseth P, Chanwanna N. Appl. Organomet. Chem. 2001; 15:
463.
9. Song ZH, Huang GL. Bull. Environ. Contam. Toxicol. 2001; 67: 368.
10. Zhihui S, Guolan H. Bull. Environ. Contam. Toxicol. 2000; 64: 723.
11. Fargasová A. Bull. Environ. Contam. Toxicol. 1996; 57: 99.
12. Bao ML, Dai SG, Pantani F. Bull. Environ. Contam. Toxicol. 1997;
59: 671.
13. Leitão MAS, Cardozo KHM, Pinto E, Colepicolo P. Arch. Environ.
Contam. Toxicol. 2003; 45: 59.
14. Gultekin F, Ozturk M, Akdogan LM. Arch. Toxicol. 2000; 74: 533.
15. Klughammer C, Heimann S, Schreiber U. Photosynth. Res. 1998;
56: 117.
16. Ouzounidou G, Moustakas M, Eleftheriou EP. Arch. Envrion.
Contam. Toxicol. 1997; 32: 154.
17. Stäb JA, Traas TP, Stroomberg G, Kesteren J, Leonards P,
Hattum B, Brinkman UATh, Cofino WP. Arch. Environ. Contam.
Toxicol. 1996; 31: 319.
18. Harino H, Fukushima M, Kawai S. Arch. Environ. Contam. Toxicol.
2000; 39: 13.
19. Looser W, Bertschi S, Fent K. Appl. Organomet. Chem. 1998; 12:
601.
20. Shim WJ, Oh JR, Kahng SH, Shim JH, Lee SH. Arch. Environ.
Contam. Toxicol. 1998; 35: 41.
21. Stäb JA, Traas TP, Stroomberg G, van Kesteren J, Leonards P, van
Hattum B, Brinkman UATh, Cofino WP. Arch. Environ. Contam.
Toxicol. 1996; 31: 319.
22. Coloso RM, Borlongan IG. Bull. Environ. Contam. Toxicol. 1999; 63:
297.
23. Mooney HM, Patching JW. J. Ind. Mirobiol. Biotechnol. 1998; 20:
200.
24. Huang GL, Song ZH, Liu GL, Zhang WH. Appl. Organomet. Chem.
2002; 16: 177.
Appl. Organometal. Chem. 2005; 19: 807–810